Successful reproduction requires faithful transmission of DNA during gamete formation and union. The DNA of the paternal gamete is packaged uniquely from that of other cell types. Sperm chromatin packaging consists of both a uniquely tight compaction of DNA by sperm-specific scaffolding molecules and the association of factors required for spermatogenesis or functions after fertilization. Sperm-specific chromatin packaging is conserved from worms to humans; however, the mechanisms used to package sperm DNA are largely unknown. Systematic dissection of factors required for sperm chromatin assembly in a genetically amenable system, such as Caenorhabditis elegans may identify vital components required for spermatogenesis in all organisms. The goal of this project is to develop methodology to identify and characterize sperm chromatin assembly factors in C. elegans. A proteomic approach is used to identify candidate sperm chromatin assembly factors. Chromatin is biochemically purified from C. elegans sperm and oocyte cells. Each sample is then subjected to specialized mass spectrometric analysis using the multidimensional protein identification technique (MudPIT). Results are analyzed against the sequenced C. elegans genome allowing identification of associated proteins. Protein profiles from sperm and oocyte samples are compared to subtract general factors and to identify proteins that are specific to highly condensed sperm DNA. The role of identified proteins in sperm chromatin assembly will be assessed by reducing function of candidate proteins. Requirements for these proteins in spermatogenesis, chromosome organization, and chromosome dynamics will be examined. Localization of candidate proteins will be used to define how and when these proteins function in chromosome assembly. The methodology using C. elegans developed in this proposal will allow rapid identification of sperm-specific factors that condense and organize chromatin. These factors may be conserved and play vital roles in fertility and reproduction in all organisms.